ASK1 Inhibits Astroglial Development via p38 Mitogen-Activated Protein Kinase and Promotes Neuronal Differentiation in Adult Hippocampus-Derived Progenitor Cells

Abstract

The mechanisms controlling differentiation and lineage specification of neural stem cells are still poorly understood, and many of the molecules involved in this process and their specific functions are yet unknown. We investigated the effect of apoptosis signal-regulating kinase 1 (ASK1) on neural stem cells by infecting adult hippocampus-derived rat progenitors with an adenovirus encoding the constitutively active form of ASK1. Following ASK1 overexpression, a significantly larger number of cells differentiated into neurons and a substantial increase in Mash1 transcription was observed. Moreover, a marked depletion of glial cells was observed, persisting even after additional treatment of ASK1-infected cultures with potent glia inducers such as leukemia inhibitory factor and bone morphogenetic protein. Analysis of the promoter for glial fibrillary acidic protein revealed that ASK1 acts as a potent inhibitor of glial-specific gene transcription. However, the signal transducers and activators of transcription 3 (STAT3)-binding site in the promoter was dispensable, while the activation of p38 mitogen-activated protein kinase was crucial for this effect, suggesting the presence of a novel mechanism for the inhibition of glial differentiation.

The hippocampus is one of the two known areas of the mammalian brain where neurogenesis persists in adulthood (3, 11, 30). Multipotent progenitor cells are located in the subgranular zone of the hippocampus, where they proliferate, survive, and migrate to the granular zone that is finally formed by terminally differentiated neurons. Differentiation of these stem cells is orchestrated by various extrinsic and heritable intrinsic factors that determine proper spatial and temporal cellular development as well as their integration into the pre-existing architecture of the dentate gyrus.

In order to study the events and stimuli that control differentiation and lineage commitment, multipotent neural stem cells can be isolated and expanded in vitro from primary cultures after serial passages in the presence of either basic fibroblast growth factor (bFGF) or epidermal growth factor (13, 17, 19, 28). Adult hippocampus-derived rat progenitors (AHPs) are bFGF-dependent, self-renewing neural progenitor cells that have been shown to give rise to all cell types found in the central nervous system, such as neurons, astrocytes, and oligodendrocytes (27). Furthermore, grafting of AHPs into neurogenic areas of the brain like the hippocampus, the rostral migratory pathway, and the retina showed the ability of these cells to adopt even nonhippocampus neuronal phenotypes dependent on the site of grafting, indicating that AHPs can not only integrate well into the host tissue but also differentiate according to surrounding environmental cues (13, 35, 38, 42).

A variety of stimuli promoting neurogenesis from neural stem cells in vivo and in vitro have been identified, such as retinoic acid, neurotrophins, steroid hormones, and insulin-like growth factor 1 (1, 5, 12, 37). Even though the exact mechanisms of action are still poorly understood, these neurogenic stimuli promote the expression of several basic helix-loop-helix transcription factors (bHLH) mediating neuronal differentiation, such as Mash1, neurogenin1 and 2, and NeuroD. Overexpression and loss-of-function studies have suggested that precise temporal and spatial expression of these bHLH transcription factors is critical for proper development of the nervous system (7, 8, 15, 34). While some members of the neural subclass of bHLH factors, such as NeuroD in postmitotic neuronal precursors, are expressed at later stages of neuronal differentiation, Mash1 and neurogenin1 are expressed in proliferating neuronal progenitors and early differentiating neurons (9, 20, 21). Astroglial differentiation, however, has been shown to be promoted by notch signaling (40) as well as by members of the gp130 group of cytokines, including, among others, leukemia inhibitory factor (LIF) and ciliary neurotrophic factor (2, 4, 17, 29). LIF activates a receptor-associated tyrosine kinase, the Janus kinase, which in turn phosphorylates STAT1 and STAT3. These cytoplasmic proteins dimerize upon phosphorylation and are translocated to the nucleus after binding the CBP/p300 coactivator complex. Once inside the nucleus, they activate transcription of glial fibrillary acidic protein (GFAP), a glial-specific intermediate filament, via the STAT1- and STAT3-binding site of the GFAP promoter. Bone morphogenetic proteins (BMPs) have been shown to be able to promote neuronal as well as astroglial differentiation in various culture systems (14, 22, 24, 43). It was recently suggested that the ability of BMPs to promote one rather than the other fate is dependent on the presence or absence of neurogenin, since neurogenin has been shown to bind and thereby sequester the CBP/p300 coactivator complex (36). Neurogenin thereby actively inhibits the glia-inducing capacities of LIF and BMPs by sequestering their coactivators away from the glia-specific promoter.

Apoptosis signal-regulating kinase 1 (ASK1) is a well conserved and ubiquitously expressed intracellular serine/threonine mitogen-activated protein (MAP) kinase kinase kinase that was first identified as a mediator of tumor necrosis factor alpha-induced apoptosis by activation of the p38 MAP kinase and the c-jun N-terminal-activating kinase pathways (16). ASK1 is involved in induction of apoptosis in response to various other cytotoxic stresses, such as UV light, Fas, or reactive oxygen species in several cell types of neuronal origin, such as sympathetic neurons and differentiated PC12 cells (18). However, recent studies of keratinocytes and undifferentiated PC12 cells showed that ASK1 could promote cellular differentiation and survival rather than apoptosis, suggesting that ASK1 might have opposing functions depending on cell type and status (33, 39).

However, the cell types used in these two studies are either nonneural cells or tumor cells of the peripheral nervous system and therefore do not provide information about the role of ASK1 in the regulation of lineage determination in endogenous multipotent neural stem cells of the central nervous system (CNS).

In the present study, we used AHPs in order to investigate the effect of ASK1 signaling on survival, proliferation, and lineage differentiation in progenitor cells of the CNS. We addressed these questions because further understanding of the role of ASK1 signaling may be of potential value for future cell-based therapeutic strategies in neurodegenerative diseases.

MATERIALS AND METHODS

Cell culture of AHPs.

AHPs were kindly provided by F. Gage (Salk Institute, La Jolla, Calif.) and maintained as described previously (26). Clonal progenitor cells were used between passages 10 and 20 postcloning. For proliferating conditions, clonal progenitor cells were cultured in Dulbecco's modified Eagle's medium-Ham's F12 (1:1) containing N2 supplement (Life Technologies), l-glutamine, and 20 ng of recombinant human bFGF per ml. When used for experiments, the cells were plated at different densities on polyornithine-laminin-coated plates in medium lacking the mitogen bFGF.

Adenoviral vectors.

The recombinant adenoviruses encoding LacZ (Ad-LacZ), the hemagglutinin (HA)-tagged kinase mutant ASK1 (Ad-ASK1-KM), and the constitutively active ASK1 (Ad-ASK1-ΔN) were described previously (23, 31, 32). The adenovirus was propagated in 293 cells to produce high-titer stocks. The cells were infected for 3 to 4 days, harvested, and lysed by five cycles of freeze-thawing. The lysate was centrifuged at 10,000 × g for 20 min at 4°C, and the stocks were kept at −80°C. Infection efficiency was judged by staining for β-galactosidase.

Growth factor treatment.

For treatment with LIF or BMP, AHPs were cultured as described and infected with Ad-ASK1-ΔN, Ad-ASK1-KM, or Ad-LacZ on days in vitro 2 (DIV 2). After another 24 h, the cells were treated with either LIF (GIBCO BRL) or BMP6 (kindly provided by Creative Biomolecules) until further analysis. Concentrations ranged from 30 to 50 ng/ml for BMP6 and from 50 to 80 ng/ml for LIF in order to induce expression of GFAP, since these concentrations have been shown to effectively induce GFAP expression in various culture systems (6, 24, 25, 36).

Treatment with SB 203580 and SB 202190.

SB 203580 and SB 202190 were purchased from Calbiochem, dissolved in dimethyl sulfoxide (DMSO) at 20 mM, and stored at −20°C. At the time of treatment, stocks of the inhibitor were thawed, prediluted in medium, and added to the well to the designated final concentration. For long-term analysis, the inhibitor was repeatedly added to the culture medium every 2 days.

Immunohistochemistry. (i) 3,3-Diaminobenzidine staining.

AHPs were cultured on chamber slides (Labtek; Nunc) and were fixed in 4% paraformaldehyde for 20 min at room temperature. After being washed with phosphate-buffered saline (PBS), the cells were quenched with 3% hydrogen peroxide for 10 min, washed again with PBS, and preincubated with Tris-buffered saline (TBS)-based blocking buffer containing 0.1% Triton X-100, 3% bovine serum albumin, and 3% normal horse serum for 1 h at room temperature. The cultures were then incubated with mouse monoclonal antimicrotubule-associated protein 2ab (Map2ab) (Sigma-Aldrich) diluted 1:1,000 in the same blocking buffer. After three washes in TBS, the cultures were incubated for 1 h at room temperature with biotinylated anti-mouse antibody (Vector Laboratories) diluted 1:200. The cells were washed, and labeling was visualized by incubation with an ABC Elite kit (Vector Laboratories) for 1 h at room temperature and finally with 0.05% 3,3-diaminobenzidine-0.03% H₂O₂ for 5 min.

(ii) Immunofluorescence.

For immunofluorescence, the cells were cultured on chamber slides and fixed for 15 min with ice-cold methanol 4 and 9 days after infection. The cells were incubated in TBS-based blocking buffer for 1 h at room temperature. After being rinsed with PBS and TBS, the cells were incubated with primary antibodies diluted in the same blocking buffer overnight at 4°C. After three washes with PBS and TBS, the cells were incubated with fluorescein isothiocyanate- or Texas red-conjugated secondary antibodies (Jackson Immunoresearch) diluted 1:100. For nuclear counterstaining, the cells were incubated in 50 ng of Hoechst 33258 (Sigma-Aldrich) per ml for 20 min before being mounted in Dako fluorescent medium (Dakoplatts AB). The following primary antibodies were used: mouse monoclonal anti-Map2ab (1:100), mouse monoclonal anti-GalC (1:100; Boehringer Mannheim), and rabbit polyclonal anti-GFAP (1:400; Dako).

Neurite outgrowth assay.

For neurite outgrowth assay, AHPs were cultured on chamber slides and infected with adenoviral constructs carrying the ASK1 kinase mutant, ASK1-KM, or the constitutively active ASK1, ASK1-ΔN, as described. After another 5 days, the cells were fixed and stained for Map2ab and observed under a standard light microscope. To determine the percentage of neurite-bearing cells, the number of Map2ab-positive cells with a process longer than one cell diameter was determined and compared to the total number of Map2ab-positive cells.

[³H]thymidine incorporation assay.

For [³H]thymidine incorporation assays, AHPs were cultured on 24-well plates as described and infected with Ad-ASK1-ΔN, Ad-ASK1-KM, or Ad-LacZ. At the time of analysis, the cells were pulsed with 0.5 μCi of [methyl-³H]thymidine (Amersham) per ml of medium for another 4 h. Then the cells were washed once with ice-cold PBS and precipitated with 5% trichloracetate on ice for 30 min. The precipitate was then dissolved in 1 M NaOH, neutralized with 1 M HCl, and transferred to vials containing 10 ml of scintillation fluid. Radioactivity was counted by using a β-counter (Wallac).

MTT assay.

The MTT assay is based on the ability of mitochondria of viable cells to reduce 3-(4,5-dimethyldiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), a light yellow compound, into a dark blue product, formazan MTT, that can be quantified spectrophotometrically. In order to assess cell survival, cells were cultured on 24-well plates as described. Sixty microliters of MTT solution (5 mg/ml; Sigma) was added to the medium, and the mixture was cultured for another 4 h at 37°C. Solubilization buffer was then added, and the mixture was cultured for another 30 min to release the formazan salt. Then absorbance was measured at 570 nm.

LDH cytotoxicity assay.

Cell death was determined by a method based on spectrophotometric measurement of lactate dehydrogenase (LDH) release by using the Cyto Tox 96 nonradioactive cyotoxicity assay kit (Promega) according to the manufacturer's protocol. For each treatment group, total LDH release was determined after addition of Triton X-100 to a final concentration of 10%. The results in Ad-ASK1-ΔN-infected wells were expressed as percentages relative to the maximal LDH release in each treatment group and compared to the ratio for the Ad-ASK1-KM-infected control.

Fluorescence activated cell sorter (FACS) analysis.

Propidium iodide (PI) staining was performed using the Cycle TEST PLUS DNA reagent kit (Becton Dickinson) according to the manufacturer's protocol. Briefly, cells were seeded on 6-well plates and infected with Ad-ASK1-ΔN or Ad-ASK1-KM on DIV 2 as described above. After 4 days of infection, the cell nuclei were isolated and stained with PI before the emitted fluorescence (25,000 nuclei/sample) was analyzed using a FACS Calibur flow cytometer (Becton Dickinson).

Luciferase reporter assay.

For luciferase reporter assays, 35,000 cells were seeded on 12-well plates as described. The human GFAP promoter (positions −1,873 to +130) and the STAT3-binding site mutated human GFAP promoter were gifts from T. Honjo (Kyoto University, Japan) and were described previously (40). The Mash1 promoter was provided by J. E. Johnson (University of Texas Southwestern Medical Center) (41), and the p38 MAP kinase expression vectors for the wild-type and dominant-negative mutant were donated by M. Landström (Ludwig Institute for Cancer Research, Uppsala, Sweden). After another 2 days, the cells were transfected with the reporter plasmids and expression vectors by using FuGENE6 (Boehringer Mannheim). The total amount of DNA was kept constant. The cells were infected 4 h after transfection with adenovirus encoding LacZ, ASK1-ΔN, or ASK1-KM as described. In the case of treatment with LIF or BMP, AHPs were treated with the growth factor 24 h after infection and a luciferase assay was performed after another 12 h. Absolute luciferase activity was standardized to the total protein amount or β-galactosidase-activity. In each experiment, the samples were analyzed in triplicate, and each experiment was repeated at least three times.

Western blotting.

For Western blotting analysis, AHPs were cultured on polyornithine-laminin-coated 24-well plates as described. At the time of harvest, the cells were washed with PBS and lysed on ice in buffer containing 150 mM NaCl, 50 mM Tris-HCl (pH 8.0), 0.5 mM EDTA, 1% NP-40, 0.5% CHAPS {3-[(3-cholamidopropyl)-dimethylammonio]-l-propanesulfonate}, and 0.1% sodium dodecyl sulfate (SDS). Phenylmethylsulphonyl fluoride and aprotinin were freshly added each time to a final concentration of 1 mM to inhibit protease activity. Cellular debris was removed by centrifugation, and the protein concentration of each sample was determined by using a DC protein assay (Bio-Rad). Equal amounts of protein were separated on SDS-10% to 12% polyacrylamide gel electrophoresis and then electroblotted onto a polyvinylidene difluoride membrane by semidry transfer (Bio-Rad). After being incubated with 5% nonfat skim milk in TBS with 0.1% Tween 20, the membranes were probed with primary antibodies. The following primary antibodies were used: rabbit polyclonal anti-GFAP (1:2,000; Dako), mouse monoclonal anti-β-III-tubulin (1:500; Sigma), mouse monoclonal antiactin (1:500; Sigma), rabbit polyclonal anti-STAT3 and anti-phospho-tyr-STAT3 (1:1,000; Cell Signaling Technology, Inc.), rabbit polyclonal antibodies against phosphorylated and total p38 MAP kinase (1:1,000; New England Biolabs, Inc.), and rabbit polyclonal anti-phospho-Smad1/5 (1:2,000; a gift from C. H. Heldin at Ludwig Institute, Uppsala, Sweden). The rat monoclonal antibody against the HA tag (3F10; Roche) was directly conjugated with horseradish peroxidase and used at a concentration of 1:500. Horseradish peroxidase-conjugated anti-rabbit and anti-mouse immunoglobulin G were used as secondary antibodies at concentrations of 1:5,000 (New England Biolabs). Signals were detected by enhanced chemiluminescence (ECL; New England Biolabs).

RT PCR.

Total RNA was extracted by the guanidinium thiocyanate method of Chomczynski and Sacchi (10). For cDNA synthesis, random hexamer primers (Roche) were used. First-strand cDNAs were synthesized from 1 μg of RNA using Moloney murine leukemia virus reverse transcriptase (RT) (Invitrogen). The PCR was carried out by using standard protocol with Taq polymerase (Fermentas). The cycling parameters were as follows: denaturation at 94°C for 4 min, followed by 25 to 28 cycles of denaturation at 94°C for 30 s, an annealing step for 45 s, and 72°C for 1 min 30 s. The last cycle consisted of a final extension step at 72°C for 6 min. The following nucleotide primers were used: Mash1 sense, 5′-GGA ACT GAT GCG CTG CAA AC, and antisense, 5′-CCT GCT TCC AAA GTC CAT TCC; and Actin sense, 5′-AAG ATG ACC CAG ATC ATG TTT GAG, and antisense, 5′-AGG AGG AGC AAT GAT CTT GAT CTT. The samples were run on 1% agarose gel containing ethidium bromide and analyzed by using a FLA 2000 plate reader (Fujifilm).

Microscopic analysis.

The cultures were analyzed by standard light and epi-illumination fluorescence microscopy, and color images were generated by using a Zeiss fluorescence microscope attached to a Nikon digital camera. The images were then processed for publication by using Adobe Photoshop (Adobe Systems).

Statistics.

All experiments were replicated at least three or four times. Overall significance was determined by submitting the data to one-way analysis of variance (ANOVA). The significance of between-group differences was determined by the Scheffé post hoc test.

RESULTS

Adenovirus-mediated ASK1 transduction yields efficient expression in AHPs.

Two days after being seeded, AHPs were infected with Ad-LacZ, Ad-ASK-KM, or Ad-ASK1-ΔN, which is constitutively active because it lacks the N-terminally located binding site for thioredoxin, the physiological inhibitor of ASK1 (32). Our adenoviral expression system provided infection efficiency up to 90% to 95%, as determined by staining for β-galactosidase (data not shown). Western blotting for the HA tag showed that expression of the infected constructs was already detectable 8 h after infection. However, the expression maximum was not reached before 1 to 2 days after infection and persisted until about 4 days after infection. Analysis of HA expression for time points later than 4 days after infection revealed a gradual decrease of overexpressed ASK1-ΔN protein (Fig. 1).

FIG. 1.

Adenovirus-mediated expression of ASK1-ΔN in AHPs. AHPs were infected with adenovirus carrying ASK1-ΔN on DIV 2 and harvested for Western blot analysis at the indicated time points.

Moderate expression of ASK1 induces differentiation of AHPs.

In order to investigate the effect of ASK1 on the growth rate and cell survival of AHPs, cells were cultured as described and infected with Ad-LacZ, Ad-ASK1-KM, or Ad-ASK1-ΔN on DIV 2. MTT assays, [³H]thymidine incorporation assays, LDH cytotoxicity assays, and FACS analysis were performed on day 2 or 4 after infection. While the MTT assay, an assay reflecting cell viability, already showed a strong decrease in cell viability 48 h after infection at a multiplicity of infection (MOI) of 100 and above, no significant reduction in cell viability was observed at MOIs of between 5 and 25 (Fig. 2A).

FIG. 2.

ASK1 differentiates AHPs. (A) AHPs were infected with adenovirus carrying LacZ, ASK1-KM, or ASK1-ΔN on DIV 2, and after another 2 days, cells were subjected to MTT assay. (B) [³H]thymidine incorporation assays were done on cells as described for panel A. (C) An LDH cytotoxicity assay was performed after 4 days of infection. (D) For FACS analysis, AHPs were cultured on 6-well plates, and PI staining was performed after 2 days of infection. The values represent means plus standard errors of the means (SEM; n = 3) from three independent experiments. An asterisk indicates a P value of <0.05, two asterisks indicate a P value of <0.01, and three asterisks indicate a P value of <0.001 in comparison to the LacZ-infected (A and B) or KM-infected (C) control. Results were determined by ANOVA and the Scheffé post hoc test.

[³H]thymidine incorporation assays, however, which reflect cell proliferation, displayed a moderate but significant decrease after 48 h of infection with Ad-ASK1-ΔN, even between MOIs of 10 and 25, compared to cell proliferation levels for Ad-LacZ- or Ad-ASK1-KM-infected control cultures (Fig. 2B). Considering that cell viability at these MOIs was unaffected, this result suggests that upon infection with Ad-ASK1-ΔN, AHPs were induced to differentiate.

The absence of significant cell death after infection with Ad-ASK1-ΔN at MOIs between 5 and 25 was confirmed by the LDH cytotoxicity assay. However, cultures infected with Ad-ASK1-ΔN at MOIs of 100 and above displayed a significant increase in LDH release compared to that of the Ad-ASK1-KM-infected control, confirming the notion that cell death was induced at these MOIs, as suggested by the MTT data (Fig. 2C).

The nuclei of Ad-ASK1-infected AHPs were stained for PI and analyzed by FACS at 4 days after infection with Ad-LacZ, Ad-ASK1-KM, or Ad-ASK1-ΔN. A significantly higher number of cells was arrested in G₂/M phase upon infection with Ad-ASK1-ΔN than for cultures infected with Ad-ASK1-KM (Fig. 2D). This result was consistently observed at MOIs between 5 and 25. Ad-LacZ-infected cultures did not differ in their staining profiles from those of Ad-ASK1-KM-infected cultures at any tested MOI (data not shown). These findings strongly suggest that growth arrest was associated with differentiation of AHPs in response to moderate levels of ASK1 overexpression.

ASK1 promotes neuronal differentiation in adult hippocampal progenitors.

In order to evaluate the effect of ASK1 on differentiation in AHPs, cells were infected with different MOIs of Ad-ASK1-ΔN at 48 h after seeding and processed for immunohistochemical staining after another 4 or 9 days. Cells were stained for Map2ab, a marker for mature neurons, in order to test the hypothesis that infection with Ad-ASK1-ΔN induces neuronal lineage determination in the progenitors. In comparison to that for Ad-LacZ- or Ad-ASK1-KM-infected cultures, the number of Map2ab-positive cells was found to be slightly increased 4 days after infection with Ad-ASK1-ΔN. However, a significant increase in Map2ab-positive cells was not observed before 8 to 9 days after Ad-ASK1-ΔN infection. This effect was already apparent at a MOI of 5 but was most prominent at the MOIs of 10 and 25, where the number of Map2ab-positive cells in Ad-ASK1-ΔN-infected cultures was increased about two- to threefold compared to that for the controls (Fig. 3A and B). Also, the number, length, and thickness of the processes of the MAP2ab-positive cells were increased in Ad-ASK1-ΔN-infected cultures compared to those of the control cultures. In order to quantify the neurite-inducing capacity of ASK1 in cultures of AHPs, we attempted to assess neurite outgrowth. AHPs were cultured on chamber slides as described, fixed, and stained for Map2ab after 5 days of infection. When neurons exhibiting one or more neurites longer than one cell in diameter were counted and related to the total number of Map2ab-positive cells, the percentage of neurite-bearing cells was increased up to 52% ± 7% in Ad-ASK1-ΔN-infected cultures, whereas only 21% ± 5% of the neurons displayed neurite outgrowth in cultures infected with Ad-ASK1-KM, suggesting that the kinase activity of ASK1 plays an important role in the induction of neurite outgrowth in AHP cultures (Fig. 3C and D).

FIG. 3.

ASK1 induces neuronal differentiation in AHPs. (A and B) AHPs were infected with adenovirus encoding LacZ, ASK1-KM, or ASK1-ΔN and processed for immunofluorescent staining for Map2ab 9 days after infection. (C and D) A neurite outgrowth assay was performed at 5 days after infection. (E) AHPs were immunoblotted for β-III-tubulin at 9 days after infection. (F) Densitometric analysis of three independent Western blots for β-III-tubulin is shown. (G) AHPs were transfected with Mash1-Luc 4 h prior to infection. Luciferase activity was determined at 36 h after transfection. (H) RT PCR analysis of AHPs was performed 2 days after infection with Ad-LacZ, Ad-ASK1-KM, or Ad-ASK1-ΔN. The values represent means + SEM (n = 3) from three independent experiments. An asterisk indicates a P value of <0.05, two asterisks indicate a P value of <0.01, and three asterisks indicate a P value of <0.001 in comparison to the LacZ-infected (B, F, and G) or KM-infected (D) control. Results were determined by ANOVA and the Scheffé post hoc test. Scale bar, 10 μM.

To further confirm the induction of neuronal differentiation by ASK1, we performed Western blot analysis in order to examine the expression of β-III-tubulin, a neuron-specific isoform of β-tubulin. As expected, we found significantly higher expression of β-III-tubulin in Ad-ASK1-ΔN-infected cultures than in Ad-LacZ- or Ad-ASK1-KM-infected cultures after infection with MOIs of between 5 and 25, indicating that the progenitor cells adopted the neuronal phenotype in response to ASK1 (Fig. 3E). Although this effect had already become apparent 4 days after infection, β-III-tubulin expression was maximal at 8 to 10 days after infection, reflecting the time needed for the progenitors to differentiate to neurons in this culture system. In order to quantify the increase in β-III-tubulin expression, we performed densitometric analysis on three independent blots where β-III-tubulin signals were normalized to the levels of β-actin present in the same sample and expressed as percentages of the control sample infected with Ad-LacZ. Protein levels of β-III-tubulin were consistently higher in cultures infected with Ad-ASK1-ΔN than in the control cultures, confirming the previous findings that overexpression of Ad-ASK1-ΔN increases the expression of neuronal markers in cultures of AHPs (Fig. 3F). Furthermore, we observed that transcription of Mash1, a proneural gene, was strongly induced in AHPs in response to Ad-ASK1-ΔN overexpression compared to that for the control cultures. When AHPs were transfected with a 1.0-kb Mash1 promoter, luciferase activity was about two to three times higher in Ad-ASK1-ΔN-infected cultures than in cultures infected with Ad-ASK1-KM or Ad-LacZ (Fig. 3G). In order to evaluate whether Ad-ASK1-ΔN overexpression actually increases endogenous Mash1 mRNA expression in cultures of AHPs, we performed RT PCR analysis. We found that the progenitors indeed displayed higher levels of mRNA for Mash1 after infection with Ad-ASK1-ΔN than did the control cultures (Fig. 3H).

ASK1 promotes neuronal differentiation in a p38-dependent manner.

Since ASK1 is an upstream kinase in many known signaling pathways, we investigated which downstream kinase might be the mediator of the ASK1-induced neuronal differentiation observed in our cultures. p38 MAP kinase has been shown to be one of the most prominent and best characterized downstream effector kinases of ASK1 in many systems. In order to evaluate whether overexpression of Ad-ASK1-ΔN was able to induce activation of the p38 MAP kinase pathway in cultures of AHPs, cells were infected with adenovirus encoding LacZ, ASK1-KM, or ASK1-ΔN on DIV 2 as described. Western blot analysis for phosphorylated p38 MAP kinase was performed after another 24 h. While phosphorylated p38 MAP kinase was clearly detectable upon overexpression of Ad-ASK1-ΔN, detectable levels of activated p38 MAP kinase were absent in Ad-LacZ- and Ad-ASK1-KM-infected cultures (Fig. 4A). When cultures were preincubated with the p38-specific inhibitor SB 203580, infected as described, and processed by immunohistochemistry for Map2ab after 9 days of infection, Ad-ASK1-ΔN-infected cultures displayed a significantly higher number of cells staining positive for Map2ab than Ad-ASK1-KM-infected cultures, a finding that is consistent with our observations described in Fig. 3. However, when Ad-ASK1-ΔN-infected cultures had been preincubated with SB 203580 prior to infection, the number of Map2ab-positive cells did not significantly differ from that for the control cultures, suggesting that inhibition of the p38 Map kinase pathway by SB 203580 abolished the Ad-ASK1-ΔN-mediated induction of neuronal differentiation in our cultures (Fig. 4B and C). Furthermore, we sought to determine whether the Ad-ASK1-ΔN-mediated induction of the Mash1 gene was dependent on p38 MAP kinase activation. Therefore, AHPs were cultured as described and transfected with a 1.0-kb Mash1 promoter on DIV 2. Prior to infection, cultures were incubated with either DMSO, SB 203580, or SB 202190, and luciferase activity was determined after another 36 h. While Mash1 luciferase activity did not significantly change upon the presence of SB 203580 or SB 202190 in cultures infected with Ad-ASK1-KM, the substantial increase in luciferase activity observed in DMSO-treated Ad-ASK1-ΔN-infected cultures was found to be almost completely abolished in the presence of either one of the two p38 MAP kinase-specific inhibitors (Fig. 4D).

FIG. 4.

ASK1-induced neuronal differentiation is dependent on p38 MAP kinase activation. (A) AHPs were infected with adenovirus carrying LacZ, ASK1-KM, or ASK1-ΔN on DIV 2 and harvested for Western blot analysis for phosphorylated p38 after another 24 h. (B) AHPs were incubated with 10 μM SB 203580 prior to infection with Ad-ASK1-KM or Ad-ASK1-ΔN. The culture medium was sup-plemented with SB 203580 every second day, and cells were processed for immunohistochemical staining for MAP2ab 9 days after infection. (C) Cell counts of Map2ab-positive cells cultured as described for panel B. (D) AHPs were transfected with Mash1-Luc and incubated with DMSO as a control or with SB 203580 or SB 202190 prior to infection with Ad-ASK1-KM or Ad-ASK1-ΔN. Luciferase activity was measured 36 h after infection. The values represent means + SEM (n = 3). Three asterisks indicate a P value of <0.001 in comparison to the KM-infected control, two number signs indicate a P value of <0.01, and three number signs indicate a P value of <0.001 in comparison to the DMSO control infected with the same viral construct. Results were determined by ANOVA and the Scheffé post hoc test. Scale bar, 10 μM.

ASK1 inhibits the development of glial cells in cultures of AHPs.

In order to determine possible effects of ASK1 on the glial cell population in the cultures, cells were infected with adenovirus encoding LacZ, ASK1-KM, or ASK1-ΔN on DIV 2 as described. After another 4 or 10 days in culture, cells were harvested, lysed, and subjected to Western blot analysis for GFAP. When examined at 4 days after infection, GFAP expression was already strongly decreased at a MOI of 5, and it decreased further in an MOI-dependent manner than did the Ad-LacZ- or Ad-ASK1-KM-infected cultures (Fig. 5A). In order to quantify this MOI-dependent decrease of GFAP expression, we performed densitometric analysis on three independent blots as described above. Already having an MOI of 5, the expression of GFAP decreased to 25% at 4 days after infection with Ad-ASK1-ΔN, decreasing further to 10% to an MOI of 50, which appeared to be the most effective MOI in this respect (Fig. 5B).

FIG. 5.

ASK1 inhibits glial differentiation in AHPs. (A) AHPs were cultured without bFGF for 2 days and then infected with adenovirus carrying LacZ, ASK1-KM, or ASK1-ΔN. After another 4 days, cells were processed for Western blot analysis. (B) Densitometric analysis of three independent Western blot assays for GFAP is shown. (C) Immunofluorescence staining for GFAP 4 days after infection. (D) Cell counts of GFAP-positive cells stained 4 and 9 days after infection. (E) AHPs were cultured for 10 days after infection and immunoblotted for GFAP. The values represent means + SEM (n = 3). Two asterisks indicate a P value of <0.01, and three asterisks indicate a P value of <0.001 in comparison to the LacZ-infected control. Results were determined by ANOVA and the post hoc Scheffé test. Scale bar, 10 μM.

Immunohistochemical staining for GFAP revealed that, indeed, the number of GFAP-positive cells was significantly decreased in Ad-ASK1-ΔN-infected cultures of AHPs compared to that for Ad-LacZ- or Ad-ASK1-KM-infected cultures after 4 days of infection (Fig. 5C). Counting of GFAP-positive cells at this time point confirmed that only 7% ± 2% of all cells were positive for GFAP in Ad-ASK1-ΔN-infected cultures, whereas in control cultures 21% ± 4% of the cells were GFAP positive (Fig. 5D). However, when cells were left in culture for 9 to 10 days, GFAP expression in Ad-ASK1-ΔN-infected cultures was increased again, even reaching baseline levels. This result was determined by immunohistochemical staining as well as by Western blotting (Fig. 5D and E).

Immunohistochemical staining for GalC, a marker for oligodendroglia, did not show any difference in the number of oligodendrocytes in cultures infected with Ad-ASK1-ΔN than in Ad-ASK1-KM-infected cultures, excluding the possibility that the progenitors adopted oligodendroglial instead of astroglial fate (data not shown).

ASK1 decreases the astroglial cell population by repressing GFAP promoter activity in AHPs.

In order to investigate whether the glia-depleting effect of ASK1 on cultures of AHPs is due to direct inhibition of GFAP promoter activity, AHPs were transfected with a luciferase vector controlled by the human GFAP (hGFAP) promoter at 4 h prior to infection with Ad-ASK1. Luciferase activity was measured 36 h after infection. Overexpression of ASK1-ΔN efficiently suppressed hGFAP promoter activity in an MOI-dependent manner compared to that for Ad-LacZ-infected cultures, suggesting that ASK1 directly targets the GFAP promoter (Fig. 6). Furthermore, it can be observed that ASK1-KM significantly increases GFAP promoter activity, suggesting the presence of endogenously activated ASK1 that might control GFAP promoter activity in native cultures of AHPs. However, no significant increase in protein levels of GFAP could be observed in cultures infected with Ad-ASK1-KM.

FIG. 6.

ASK1 signaling inhibits GFAP promoter activity in AHPs. AHPs were infected on DIV 2 with adenovirus carrying LacZ, ASK1-KM, or ASK1-ΔN and transfected with hGFAP-Luc 4 h prior to infection. Luciferase activity was measured after another 36 h. The values represent the means + SEM (n = 4) from one of three independent experiments. Three asterisks indicate P values of <0.001 in comparison to the LacZ-infected control. Results were determined by ANOVA and the Scheffé post hoc test.

ASK1 acts as an efficient inhibitor of glial cell development even in the presence of the glia-instructive signals of LIF and BMP.

LIF and BMP are known to be strong inducers of GFAP in progenitor cells of various origins by acting instructively on the determination of astroglial cell fate. In order to determine whether the decrease of GFAP expression in AHPs cultures after infection with Ad-ASK1-ΔN can be restored by glia-inducing signals, LIF or BMP6 was added to the cultures at 24 h after infection at a concentration of 50 ng/ml and 30 ng/ml, respectively. Western blot analysis for GFAP was performed after 3 more days. As expected, LIF and BMP6 induced a strong increase in GFAP expression in control cultures infected with Ad-ASK1-KM. However, when added to Ad-ASK1-ΔN-overexpressing cultures, LIF and BMP6 were not potent enough to increase GFAP to baseline levels, unlike the untreated culture infected with Ad-ASK1-KM, even though GFAP expression was slightly stronger than in untreated cultures infected with Ad-ASK1-ΔN (Fig. 7A). We did not observe any differences in GFAP expression between Ad-LacZ- and Ad-ASK1-KM-infected cultures treated with the cytokines. This finding suggests that even strong glia inducers, such as LIF and BMP, are not able to restore GFAP levels in cultures that are depleted of GFAP-positive cells by ASK1 to the level observed in the control cultures. Densitometric analysis of three Western blots from separate experiments confirmed this observation. Hence, LIF and BMP6 are able to increase GFAP expression in the presence of Ad-ASK1-ΔN but to only a limited extent (Fig. 7B).

FIG. 7.

Effect of ASK1 on GFAP expression in the presence of BMP and LIF. (A) AHPs were infected with Ad-ASK1-KM or Ad-ASK1-ΔN on DIV 2 and treated with 50 ng of LIF or 30 ng of BMP6 per ml, respectively, after another 24 h. GFAP expression was determined by Western blot analysis after another 3 days. (B) Densitometric analysis of three independent Western blots. (C) Cell counts of GFAP-positive cells infected and treated as described for panel A. (D) AHPs were transfected with hGFAP-Luc 4 h prior to infection and exposed to 80 ng of LIF or 50 ng of BMP6 per ml after another 24 h. Luciferase activity was determined 12 h after addition of the growth factor. Thevalues represent means + SEM (n = 3). One asterisk indicates a P value of <0.05, two asterisks indicate a P value of <0.01, and three asterisks indicate a P value of <0.001 in comparison to the buffer-treated control. One number sign indicates a P value of <0.05, two number signs indicate a P value of <0.01, and three number signs indicate a P value of <0.001 in comparison to KM-infected (B and C) or LacZ-infected (D) controls treated with the same growth factor. Results were determined by ANOVA and the Scheffé post hoc test.

In order to determine the numbers of GFAP-positive cells upon exposure to LIF and BMP, AHPs were infected on DIV 2 as described and treated with one of the cytokines at 24 h after infection. After another 3 days, cells were fixed and processed for immunohistochemical staining for GFAP. While 22% ± 3% of the cells stained positive for GFAP in untreated Ad-ASK1-KM-infected cultures, addition of 50 ng of LIF or 30 ng of BMP per ml resulted in an increase of GFAP-positive cells up to 52% ± 1% and 38% ± 2%. When Ad-ASK1-ΔN-infected cultures were left untreated, the number of GFAP-positive cells was reduced to 9% ± 2%, a phenomenon extensively described in Fig. 5. However, when LIF or BMP was added to Ad-ASK1-ΔN-overexpressing cultures, these cytokines were hardly able to elevate the numbers of GFAP-positive cells to baseline levels, unlike the results for untreated cultures infected with Ad-ASK1-KM. This finding gives further support to the hypothesis suggesting that ASK1 is an important and potent inhibitor of glial cell development (Fig. 7C).

Furthermore, we examined whether LIF and BMP6 had similar effects on the GFAP promoter activity in Ad-ASK1-ΔN-infected progenitor cultures. Cells were transfected with hGFAP promoter luciferase plasmid 4 h prior to infection with Ad-ASK1-ΔN and treated with 80 ng of LIF or 50 ng of BMP6 per ml after another 24 h. Luciferase activity was then measured at 12 h after addition of the growth factor. As already shown in Fig. 4, ASK1-ΔN induced an MOI-dependent decrease in GFAP promoter activity of up to 20% to 25% at an MOI of 25 compared to that for untreated Ad-LacZ-infected cultures. Treatment with LIF or BMP increased GFAP promoter activity significantly in Ad-LacZ- and Ad-ASK1-KM-infected wells as well as in cultures infected with Ad-ASK1-ΔN at an MOI of 5. However, at an MOI of 25, LIF and BMP6 failed to significantly increase GFAP levels in Ad-ASK1-ΔN-infected cultures. Compared to the LIF- and BMP-treated controls, Ad-ASK1-ΔN-infected cultures that had received LIF or BMP treatment consistently showed a significant decrease in GFAP promoter activity (Fig. 7D). Interestingly, when comparing the potency of the applied growth factors, we found that LIF appeared to be more potent than BMP6 in increasing protein levels of GFAP, whereas on the level of GFAP promoter activity, BMP6 seemed to be more effective. Taken together, these data show that the strong reduction of GFAP protein levels in Ad-ASK1-ΔN-infected cultures of AHPs is due to direct inhibition of GFAP promoter activity. This reduction could not be restored by instructive glia-inducing signals, such as LIF and BMP, either on the level of promoter activity or on the protein level.

ASK1 decreases GFAP protein in cultures of AHPs independently of the STAT3 pathway.

In order to investigate whether the inhibition of GFAP promoter activity by ASK1 is dependent on the STAT3-binding site of the promoter or whether ASK1 exerts its action by interfering with known upstream pathways of the STAT3-binding site, we transfected AHPs with a STAT3-binding site mutant hGFAP luciferase construct (STAT3mut-Luc), disrupting the binding of STAT3 to the GFAP promoter, and measured luciferase activity 36 h after infection with Ad-ASK1. Ad-LacZ-infected cultures transfected with STAT3mut-Luc showed a significant decrease in luciferase activity compared to that for cultures transfected with the native hGFAP promoter, suggesting that glial cell development in AHPs is dependent on STAT3 activation by endogenous signals. However, ASK1-ΔN was still able to repress the activity of the STAT3mut-Luc with the same efficiency as in cultures transfected with the native hGFAP-Luc, suggesting that negative regulation of the GFAP promoter by ASK1 is independent of the STAT3-binding site (Fig. 8A). Furthermore, we investigated whether the levels of phosphorylated Smad and tyrosine-phosphorylated STAT3 were changed in response to ASK1-ΔN overexpression. As expected, no changes in phosphorylation levels of either STAT3 or Smad were found, further supporting the finding that ASK1-mediated inhibition of GFAP promoter activity is indeed independent of activation of the STAT3-binding site in the GFAP promoter (Fig. 8B and C).

FIG. 8.

ASK1 inhibits GFAP promoter activity independently of STAT3 activation. (A) AHPs were transfected with hGFAP-Luc or a STAT3-mutated hGFAP-Luc 4 h prior to infection with Ad-LacZ, Ad-ASK1-KM, or Ad-ASK1-ΔN. Luciferase activity was determined after another 36 h. (B) AHPs were treated with 80 ng of LIF or 50 ng of BMP6 per ml at 24 h after infection with ASK1-KM or ASK1-ΔN, respectively, and cells were harvested for Western blot analysis for phospho-tyr-STAT3 20 min after addition of the growth factor. (C) Analysis of Smad phosphorylation performed as described for panel B. The values represent means + SEM (n = 4). An asterisk indicates a P value of <0.05, two asterisks indicate a P value of <0.01 in comparison to the LacZ-infected control, and a number sign indicates a P value of <0.05 in comparison to the hGFAP-Luc-transfected control infected with the same construct. Results were determined by ANOVA and the Scheffé post hoc test.

Repression of GFAP promoter activity by ASK1 is mediated via the p38 MAP kinase pathway.

In order to determine whether p38 MAP kinase signaling is involved in mediating GFAP promoter repression in AHPs by ASK1, cells were transfected with hGFAP-Luc and infected with Ad-LacZ, Ad-ASK1-KM, or Ad-ASK1-ΔN as described before. They were then treated with 10 μM SB 203580 at the time of infection, and luciferase activity was determined after another 36 h. Figure 9A shows that treatment with the p38 MAP kinase inhibitor did not change luciferase activity in Ad-LacZ- or Ad-ASK1-KM-infected cultures. In contrast, GFAP promoter activity was significantly increased in Ad-ASK1-ΔN-infected cultures after treatment with SB 203580 compared to that for Ad-ASK1-ΔN-infected cultures that received DMSO as a control. This finding suggests that the ASK1-induced repression of the GFAP promoter is indeed mediated via the p38 MAP kinase. In order to confirm this finding, we overexpressed ASK1-ΔN and p38 dominant negative (p38DN) and cotransfected with hGFAP-Luc. Luciferase activity was analyzed after another 36 h as described before. We observed that cotransfection with p38DN significantly inhibited the repressive effect of ASK1 on the GFAP promoter compared to that for ASK1-ΔN-transfected cultures that were cotransfected with either empty vector or the p38 wild type (Fig. 9B). In order to determine whether inhibition of the p38 MAP kinase pathway prior to overexpression of Ad-ASK1-ΔN would restore protein levels of GFAP, Western blot analysis for GFAP was performed on Ad-LacZ- and Ad-ASK1-ΔN-infected cultures that had been preincubated, respectively, with either DMSO as a control or with 10 μM SB 203580. Figure 9C shows that GFAP protein levels can indeed be restored upon overexpression of Ad-ASK1-ΔN when SB 203580 is present, while DMSO-treated control cultures infected with Ad-ASK1-ΔN show the already described strong reduction in GFAP expression. These data strongly suggest that ASK1-induced inhibition of GFAP expression is mediated via the p38 MAP kinase pathway.

FIG. 9.

ASK1-mediated inhibition of GFAP expression is dependent on p38 MAP kinase. (A) AHPs were transfected with hGFAP-Luc and infected with Ad-LacZ, Ad-ASK1-KM, or Ad-ASK1-ΔN 4 h after transfection. Cells were treated with 10 μM SB 202580 or DMSO as a control at the time of infection, and luciferase activity was determined after another 36 h. (B) GFAP promoter analysis on AHPs transfected with plasmids encoding either ASK1-KM or ASK1-ΔN as well as with plasmids carrying pcDNA3, p38 wild type (p38wt), or p38DN. Luciferase activity was measured 36 h after transfection. (C) AHPs were incubated with 10 μM SB 203580 prior to infection with Ad-LacZ or Ad-ASK1-ΔN. SB 203580 was readded to the culture medium 2 days after infection, and cells were harvested for Western blot analysis for GFAP after another 2 days. The values represent means + SEM (n = 3). An asterisk indicates a P value of <0.05,and three asterisks indicate a P value of <0.001 in comparison to the LacZ-infected (A) or pcDNA-transfected (B) control; two number signs indicate a P value of <0.01, and three number signs indicate a P value of <0.001 in comparison to the DMSO control infected with the same viral construct (A) or to the KM-transfected control (B). Results were determined by ANOVA and the Scheffé post hoc test.

DISCUSSION

The role of ASK1 in cell fate specification.

We have investigated the role of ASK1 in survival, proliferation, and differentiation of CNS stem cells by using bFGF-dependent multipotent neural progenitor cells derived from the adult rat hippocampus. Our data show that after infection with Ad-ASK1-ΔN a significantly higher number of cells displayed neuronal features compared to that for control cultures. Even though our cultures contained a significant number of immature neural progenitor cells, the presence of already predifferentiated precursors of the neuronal or the glial lineage at the time of infection cannot be excluded. It therefore remains to be investigated whether the increased number of neurons after infection with Ad-ASK1-ΔN is due to an induction of neuronal fate in neural stem cells or to promotion of neuronal differentiation in already predifferentiated precursors. In order to determine which of the different populations of precursors respond to ASK1, it would be necessary to distinguish between them and analyze their responses to ASK1 separately. However, determining the single differentiation stages between an immature neural stem cell and a terminally differentiated neuron is rather difficult, mainly due to the limited availability of appropriate markers to distinguish these different developmental stages. Nonetheless, we observed a significant increase in Mash1 promoter activity in response to ASK1-ΔN in the progenitor cultures. This finding indicates that there is a reason to believe that a substantial number of yet uncommitted progenitors in the culture respond to ASK1 signaling by differentiating to the neuronal lineage, since Mash1 is one of the earliest markers expressed in committed neural progenitors.

There have been no previous investigations about the role of ASK1 in glial-cell development. However, our results indicate that ASK1 may play an important role in negative regulation of glial-cell development in cultures of adult neural progenitor cells. Extrinsic and intrinsic factors that influence the fate of uncommitted multipotent progenitor cells can exert their action in two distinct ways. They can either instruct the progenitors to commit to a particular lineage, or they can allow the cells to be more susceptible to other factors determining proliferation, survival, and lineage restriction. In order to define the strength of ASK1-mediated inhibition of glial-cell development, we challenged the effect of ASK1 by simultaneous application of LIF or BMP, two known instructive inducers of astroglial cell fate. We showed that the inhibition of glial-cell differentiation by ASK1 is effective and robust, since neither of the two applied factors was able to restore either the expression levels of GFAP protein or the number of GFAP-positive cells back to baseline levels. The amount of cell death was not significantly elevated in Ad-ASK1-ΔN-infected cultures at the MOIs used for analysis of GFAP expression, indicating that the reduction in GFAP protein was unlikely to be due to cell death of GFAP-positive cells or astroglial precursors in our cultures. Furthermore, the decrease of GFAP promoter activity in response to ASK1 signaling supports the conclusion that the reduction in the number of GFAP-positive cells is indeed due to an inhibition of glial-cell development rather than to cell death.

However, when infected cells were left in culture for longer periods, the initially observed absence of GFAP-positive cells after infection with Ad-ASK1-ΔN was no longer apparent. We considered two different scenarios to explain this phenomenon. Since the infection efficiency of our adenoviral transduction is below 100%, it might be possible that the population of initially uninfected cells expanded in comparison to the population of infected cells in the course of the culture. These cells could then develop into astroglial cells and thereby replace those cells within the population of infected cells that were initially meant to adopt glial fate. Even though we cannot completely exclude this possibility, we consider it unlikely that the population of initially uninfected cells can expand in such a tremendous way, especially since our infection efficiency was as high as 90% to 95% and would therefore leave a rather small number of cells uninfected. It seems more likely that glial differentiation in neural stem cells or early glial precursors is inhibited by ASK1 as long as the adenoviral infection is able to provide a sufficient expression of ASK1-ΔN protein in the cells. Since adenoviral DNA, in contrast to retroviral DNA, does not integrate into the genome of the infected cell, the number of virus particles in each cell decreases with every cell division. Thus, the levels of ASK1-ΔN protein decrease in the course of the culture, and the inhibitory effect of ASK1 on glial differentiation in neural stem cells or early glial precursors will decrease or even be abolished as soon as ASK1-ΔN protein levels drop beneath the threshold necessary to efficiently inhibit glial differentiation. In such a case, the thereby disinhibited cells would be able to resume their development into astroglial cells and the levels of GFAP protein would not display any significant reduction compared to those of the control cultures.

Mechanism of ASK1-mediated inhibition of glial-cell development.

LIF and BMP are known to be instructive inducers of astroglial cell fate in various systems. Since BMPs have also been reported to promote neuronal differentiation, it was recently suggested that the amounts of endogenous neurogenin in each cell determine the response of the cell to BMP signaling (36). When levels of neurogenin are low, BMP induces astroglial fate in immature neural progenitor cells. In our culture system, LIF and BMP appeared to be very potent inducers of astroglial fate, suggesting that levels of endogenous neurogenin are rather low. Nonetheless, it would be possible that the effect of ASK1 on AHPs described in this study is mediated via induction or activation of endogenous neurogenin, since the effects of ASK1 on AHPs described in our study somewhat resemble the effects of neurogenin on stem cells reported elsewhere (36). However, it seems unlikely that neurogenin acts as a direct downstream mediator of ASK1 signaling in AHPs. It is well known that instructive glia-inducing signals, such as LIF, CNTF, and BMP, activate the GFAP promoter via the binding site for STAT3, which is one of the major cytoplasmic signaling molecules activated by LIF and CNTF. BMPs exert their action by phosphorylation of Smads, forming a complex with CBP/p300. This complex in turn binds to STAT3 and is thereby sequestered to the STAT3-binding site of the GFAP promoter, activating the promoter.

In our study, however, we found that the STAT3-binding site of the GFAP promoter was dispensable for the ASK1 effect, and we could not detect any changes in the phosphorylation status of STAT3 or Smad. This finding might present a yet unknown mechanism of inhibition of glial-cell development that is independent from the mechanism previously described for neurogenin.

The p38 MAP kinase pathway and its implications in cellular development.

Numerous factors and ligands have been identified that promote neuronal and/or inhibit glial differentiation of neural stem cells; however, the intracellular signaling mechanisms and their effector molecules are still unknown for most of these factors. We suggest herein a novel mechanism by which the MAP kinase kinase kinase ASK1 induces neuronal differentiation in neural stem cells and, in parallel, effectively inhibits gliogenesis via activation of the p38 MAP kinase pathway. In a previous paper, Takeda et al. reported that induction of survival and neuronal differentiation in undifferentiated PC12 cells by ASK1 is dependent on activation of the p38 MAP kinase pathway (39). However, progenitor cells of the peripheral and central nervous systems have quite different properties. Therefore, the p38 MAP kinase-mediated induction of neuronal differentiation by ASK1 could be restricted to cells of the peripheral nervous system and might not necessarily be involved in neuronal differentiation of progenitor cells derived from the CNS. Nonetheless, our data suggest that the ASK1-p38-MAP kinase pathway is an important signaling pathway for neuronal differentiation of progenitor cells derived from the CNS. Furthermore, our results indicate that astroglial development is effectively inhibited in adult neural progenitor cells when the p38 MAP kinase is activated following ASK1 signaling. It is reasonable to assume that, throughout development, once a certain cellular fate is promoted, the opposing fate would be simultaneously inhibited. We suggest herein that ASK1 exerts via the p38 MAP kinase pathway a dual role in development of immature cells of neural origin by promoting neuronal fate on the one hand and by inhibiting astroglial fate on the other.

Possible implications for ASK1 in gene therapy of neurological diseases.

Stem cells are presently believed to be a promising cell source for cell replacement strategies in neurodegenerative diseases such as Parkinson's disease. However, due to poor survival rate and a low yield of cells adopting neuronal phenotypes, neurotrophic factor delivery and genetic engineering of the cells prior to grafting are presently being investigated in order to overcome these obstacles. Astroglial differentiation within the graft may be problematic, especially when the cells are grafted into nonneurogenic areas such as the striatum. Thus, tools preventing glial differentiation in the grafted cells are of special interest. Our results indicate that genetic engineering of stem cells with ASK1 prior to transplantation might be a new strategy to improve the yield of developing neurons. Since the ability of ASK1 to inhibit glial fate appears to be the major effect on AHPs, one might consider combining ASK1 with another gene or trophic factor that instructively induces neuronal fate within the graft. Nonetheless, because a permanent ASK1 signaling in the transduced cells is necessary at least for the inhibition of glial-cell development, we suggest the use of a system in which the expression levels of ASK1 remain stable, such as a retroviral infection system.